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Abstract:

A method for treatment of a disease caused by aggregation of misfolded
proteins including subjecting a body fluid of a patient to a separation
of an aggregated misfolded protein which includes contacting both the
misfolded and normal protein with a conjugated polyelectrolyte (CPE) and
separating the CPE/protein complex from the other constituents of the
sample.

Claims:

1. A method for treatment of a disease caused by aggregation of misfolded
proteins comprising subjecting a body fluid of a patient to a separation
of an aggregated misfolded protein comprising contacting both said
misfolded and normal protein with a conjugated polyelectrolyte (CPE) and
separating the CPE/protein complex from the other constituents of the
body fluid.

6. The method according to claim 1, wherein the method is performed in
vitro.

7. The method according to claim 1, wherein the method is performed in
vivo.

8. The method according to claim 1, wherein the CPE is bound to a solid
support.

9. The method according to claim 1, further comprising detection of the
CPE/protein complex.

10. The method according to claim 1, wherein binding between CPE and
misfolded protein and detection of the CPE/protein complex is performed
simultaneously.

11. The method according to claim 9, wherein the CPE/protein complex and
the detection means are on separate sides of a barrier, such as a tube
wall, a membrane or skin.

12. A method for treatment of a disease caused by aggregation of misfolded
proteins comprising administering to a patient suffering from said
disease an amount of a CPE effective to inhibit further aggregation of
misfolded protein or to bind intermediate forms of misfolded proteins and
removing these from further reactions.

Description:

FIELD OF THE INVENTION

[0001]The present invention relates to the use of conjugated
polyelectrolytes for specific, optionally under selective conditions,
separation, for example capture, of misfolded, pathological or rogue
forms of proteins, i.e. amyloid form, misfolded form or aggregated form,
and capture of proteins which are similarly causing aggregation of
abnormal forms of proteins which in their normal form are not aggregated.
The present invention also relates to a one step method of separating and
at the same time detecting the misfolded, pathological or rogue forms of
proteins. A further embodiment of the present invention relates to the
use of conjugated polyelectrolytes as novel therapeutic agents by
interfering with the formation of, or by capturing misfolded,
pathological or rogue forms of proteins in vivo.

BACKGROUND OF THE INVENTION

[0002]The development of materials and molecules that are capable of
selectively capturing misfolded or aggregated forms of proteins,
especially the amyloid forms and pathological forms, have received a
great deal of attention, owing to their potential for being used as
analytic tools in clinical chemistry, in diagnosis, as well as
therapeutic agents. Amyloid fibrils are normally stained with small
molecule dyes, such as Congo red and thioflavin T. The great drawback of
such molecular dyes is that it is not possible to separate, i.e. bind,
capture or isolate, the misfolded protein and at the same time detect it.

[0003]One methodology for assaying for the presence of amyloid, misfolded
or pathological forms of proteins is to subject a sample to proteolysis,
for example with proteinase K, for a period sufficient to destroy the
native proteins and then perform an immunoassay using an antibody which
is not selective for amyloid, misfolded or pathological forms of proteins
in the presence of native proteins to determine the presence of said
misfolded forms of proteins. If a protease is used to remove native
proteins in a sample containing amyloid, misfolded or pathological forms
of proteins, this prevents the use of an antibody as a capture agent or
as a detection agent during the proteolysis step, since the antibody
would naturally be destroyed in the presence of the protease. Thus the
protease must be removed or deactivated before the antibody can be
introduced. It would be a great advantage to circumvent this limitation
on the procedure when capturing and detecting amyloid, misfolded or
pathological forms of proteins in the presence of native proteins.

[0004]Natural biopolymers, such as proteins, frequently have ordered
conformations, such as alpha-helix and beta-sheets, which contribute to
the three-dimensional ordered structure and the specific function of the
biopolymer. The structure of a protein is essential for the protein's
function; it has been shown by many scientists that an unfolded protein
may not be functional. More important, in the last few years there is
increasing awareness of the danger of protein misfolding and misassembly
into for example amyloid and other pathological forms. Misfolding can
change a protein from something that is useful into nonfunctional,
harmful or even toxic. Human health relies on properly folded protein,
and in vivo deposition of amyloid fibrils is associated with many
diseases of protein conformation, including Alzheimer's disease,
Huntington's disease, systemic amyloidoses, and the prion diseases. The
prion diseases, i.e. transmissible spongiform encephalopathy (TSE), in
animals [e.g. bovine spongiform encephalopathy (BSE), Scrapie and chronic
wasting disease (CWD)] and in humans [Creutzfeldt Jakob disease (CJD),
Gerstmann-Straussler-Scheinker disease (GSS), Kuru] are associated with
the conformational conversion of the normal cellular prion protein,
(PrPC), to an infectious pathogenic disease-associated isoform
denoted PrPSc. Proteins frequently alter their conformation due to
different external stimuli and the importance of conformational changes
of proteins leading to pathogenic states has been well documented.
Especially under conditions that destabilize the native state, proteins
can aggregate into characteristic fibrillar assemblies, known as amyloid
fibrils. These beta-sheet rich protein assemblies have distinctively
different conformations to that of the native state. The misfolded prion
protein is even self-propagating (infectious), a property which is
entirely encoded within the misfolded conformation. The underlying
mechanism of protein misfolding and subsequent amyloid formation is
poorly understood. Many lines of evidence support the existence of
smaller oligomeric species as intermediates on the pathway from misfolded
protein to amyloid. These oligomers vary in morphology, and only a subset
of these may be responsible for the cellular toxicity associated with
amyloid disease. Furthermore, a single soluble protein can give rise to
different "strains" of misfolded product, as evidenced by the yeast
prion, Sup35. It has been shown that the infectivity of different yeast
prion strains is dependent on the conformation of the infectious protein,
and that a single protein can adopt multiple, self-propagating
(infectious) conformations. These conformational differences underlie
heritable differences in prion strains. In addition, prion strains might
also have a major role in determining the specificity of prion
transmission.

[0005]Chronic human diseases seriously affect the healthcare system. It is
well recognized that rapid and accurate diagnostic tools are necessary to
afford early intervention and therapy. Only symptomatic therapy is
available, like in Alzheimer's disease for example, and these have
limited therapeutic efficacy. Presently there are no antemortem molecular
diagnostic tests of Alzheimer's disease or transmissible spongiform
encephalopathies (TSEs), and the clinical diagnostics that are performed
require that disease progression is severe. Further, there are no
efficient treatments available yet, and immunotherapy in for example
Alzheimer's disease holds great promise. The lack of reliable methods to
capture misfolded proteins, monitoring both treatment and disease
progression is however a severe shortcoming in treatment of most protein
misfolding related diseases.

[0006]Hence, there is a need for simple, sensitive and versatile tools
that can separate, for example capture, amyloidogenic proteins, such as
misfolded prion proteins and different strains of prion proteins, from a
sample. There is also a need for novel therapeutic agents by interfering,
binding or capturing misfolded or amyloid proteins in vivo.

[0008]Simple methods with improved affinity for capturing misfolded or
aggregated forms of proteins, especially the amyloid forms and
pathological forms, are required, especially for separating misfolded
proteins from samples (ex vivo or in vivo). Methods based on conjugated
polyelectrolytes that can capture agents for self-assembled/aggregated
forms of proteins, especially misfolded or aggregated forms of proteins,
and at the same time acting as transducer reporting the capture event in
optical signals, is therefore described in the present invention.

[0009]Moreover, the present invention also relates to the use of
conjugated polyelectrolytes as novel therapeutic agents which act by
interfering, binding or capturing misfolded, amyloid aggregated or
pathological protein forms, such as Amyloid beta in Alzheimer's or
misfolded prion proteins in TSEs. Conjugated polyelectrolytes bind and
capture these protein forms and therefore CPEs influence pathogenesis in
living organisms by acting as pharmacophores, i.e. they are therapeutic
agents. Conjugated polyelectrolytes that cross the blood brain barrier
have an effect on diseases that affect the brain, this include, but is
not limited to, Abeta amyloid pathology in living organisms, i.e.
influence Alzheimer's disease pathogenesis by acting as therapeutic
pharmacophores.

[0010]The present invention relates to the use of conjugated
polyelectrolytes (CPEs), either localized on a solid support or free in
solution, for the specific separation, for example capture, of misfolded,
pathological or rogue forms of proteins, i.e. amyloid form, misfolded
form or aggregated form, and proteins which are similarly aggregating
abnormal forms of proteins which in their normal form are not aggregated.
Said capture of misfolded proteins using a CPE or several CPEs can
optionally be performed under selective conditions for the misfolded
protein form. The present invention also relates to a one step method of
capturing and detecting, either performed selective under non-selective
conditions for misfolded proteins, of the misfolded, pathological or
rogue forms of proteins.

[0011]A further aspect of the present invention is CPEs as tools for
in-vivo imaging of misfolded or aggregated forms of proteins, especially
the amyloid forms and pathological forms. The invention also relates to
in vivo imaging methods, by for example MRI, of misfolded proteins using
functionalized conjugated polyelectrolytes.

[0012]The present invention solves the shortcomings of the prior art by
using CPEs for separation, for example capturing, binding or interfering,
of the misfolded proteins in a sample (ex vivo or in vivo), capture and
detection of the same, novel optical methods for diagnosis and potential
therapeutic agents, all based on conjugated polyelectrolytes. Prior art
have demonstrated that CPEs reliably detect amyloid fibrillation in vitro
and in ex vivo tissue samples [WO2005109005]. The physico chemical reason
for capture of amyloidogenic structures using CPEs rely on the repetitive
structure of the polyelectrolytes that associated with symmetric
repetitive molecular targets (amyloidotic molecules), providing for
extremely high affinity. Thus, the selective CPE based methods provides
for capture of pathogenic and amyloid proteins associated with a number
of diseases. Tools and methods for capture of misfolded or aggregated
forms of proteins, especially the amyloid forms and pathological forms,
but also therapies of diseases related with these proteins forms are all
based on conjugated polyelectrolytes, according to the present invention.

[0014]One aspect of the invention is the use of a conjugated
polyelectrolyte as a capture agent of misassembled/aggregated forms of
proteins, especially of amyloid fibrils and pathogenic forms of proteins.
The conjugated polyelectrolyte can either be localized on a solid
support, on a bead of any size, in a tube, in dialysis equipment, in
pumps or be provided free in solution when used as an agent to capture
misfolded proteins. The CPE can also have the form of a hydrogel. If the
CPE is provided free in solution any means to separate the misfolded
protein after being captured by the CPE can be applied, for example
separation by sedimentation, centrifugation, adsorption, absorption,
drying, boiling or evaporation. Optionally the CPE can be functionalized
to provide for means of detection, immobilization, facilitate capture,
enhanced selectivity, enhanced affinity or other features of significance
for the assay.

[0015]Interestingly, conjugated polyelectrolytes, such as poly(thiophene),
Poly(3,4-ethylenedioxythiophene) or poly(pyrrole), can be used to detect
misfolded proteins [WO2005109005]. Sensors based on conjugated
polyelectrolytes are sensitive to very minor perturbations, due to
amplification by a collective system response and therefore offer a key
advantage compared to molecular based sensors of the prior art and the
polyelectrolytes also offers a direct detection of the pathogenic prion
protein. Combine this direct detection functionality of CPEs with the
unique ability to capture misfolded proteins, according to the present
invention, and a huge advantage compared to traditional
immunohistological techniques is provided as these methods often require
the use of a primary antibody for capture and secondary antibody for
visualization of the misfolded protein. The possibility to use conjugated
polyelectrolytes as capture agents for misfolded proteins requires that
polymers are compatible with an aqueous environment and this has been
accomplished by making conjugated luminescent polyelectrolytes

[0016]A novel way to capture misfolded proteins using CPEs is described in
the present invention. Separation of misfolded proteins in a sample, for
example to remove them from said sample, can be achieved using CPEs and
has never been described before. A method to use CPEs to stain and
capture a misfolded protein in a sample solution followed by
sedimentation is provided. The methods using CPEs have other properties
than other amyloidotrophic dyes such as congo red and ThT, most
importantly CPEs provide for capture of misfolded proteins. Therefore,
several methods using CPEs to capture misfolded proteins are envisioned
in the present invention. The CPE can be immobilized on a solid support
and thereby provide a way to capture the misfolded protein to the solid
support with high selectivity and high affinity, and thereby isolate or
remove it from a sample.

[0017]In a further aspect of the invention there is provided methods for
capturing misfolded proteins, comprising exposing a conjugated
polyelectrolyte as defined above, to a sample, whereby said conjugated
polyelectrolyte selectively captures misfolded proteins in the sample.

[0018]In one aspect the present invention relates to a method for
capturing the misfolded protein species in a sample on a solid support
comprising the steps [0019]Immobilizing at least one conjugated
polyelectrolyte (CPE) on a solid support [0020]bringing the sample in
contact with the CPE [0021]optionally adding competition agents
[0022]removing the solid support from the sample or separating the solid
support from the sample, or [0023]washing the solid support without
removing the captured misfolded protein from the CPE phase
[0024]optionally detecting captured misfolded by a preferred method
whereby the original sample solution then contains none or less misfolded
proteins.

[0025]A further aspect the present invention relates to a method for
capturing the misfolded protein species in a sample in solution
comprising the steps [0026]bringing the sample in contact with at least
one conjugated polyelectrolyte (CPE) [0027]optionally adding competition
agents [0028]separation of the captured misfolded protein from the sample
[0029]optionally detecting captured misfolded proteins by a preferred
methodwhereby the original sample solution then contains none or less
misfolded proteins.

[0030]In a further embodiment of the invention, competition agents are
added to the solution to increase differentiation of CPE capturing
misfolded proteins from CPE that bind, for example unspecific binding, to
native or unfolded proteins. Such agents include, but are not limited to,
detergents, ions, salts, chelators and solvents.

[0031]If detection of misfolded protein captured by CPE is performed, the
radiation used in the method of the present invention has wavelengths in
the range from about 100 nm to about 2000 nm. In one embodiment,
radiation in the visible range is used. It is also possible to use
multiple-photon excitation, such that instead of excitation radiation of
x nm, a radiation of 2x or 3x (two-photon and three-photon excitation,
respectively) is used.

[0032]In a further aspect the invention relates to a device for performing
the methods according to the invention. Such a device is equipped with
means for bringing the CPE, either localized on a solid support or in
solution, in contact with the sample, means for optionally adding
competition agents, means for optionally removing the CPE from the sample
or separating the CPE from the sample, optionally washing the CPE phase
without removing the captured misfolded protein from the CPE phase,
optionally detecting captured misfolded by a preferred method as well as
means for optionally collecting the sample solution exposed to the CPE.

[0033]Preferably the conjugated polyelectrolyte used in the present
invention comprises copolymers or homopolymers of thiophene, pyrrole,
aniline, furan, phenylene, vinylene, fluorene, ethylenedioxythiophene or
their substituted forms, and the conjugated polyelectrolyte may have one
or more ionic side chain functionalities, such as amino acids, amino acid
derivatives, neurotransmittors, monosaccharides, nucleic acids, or
combinations and chemically modified derivatives thereof. The ionic
functionalities may comprise one or more anionic and cationic side chain
functionalities.

[0036]In one aspect of the invention there is provided a method of
detecting the separation of misassembled/aggregated forms of proteins,
especially the formation of amyloid fibrils and pathogenic forms of
proteins, from natural forms of proteins behind a barrier such as a tube
wall, a membrane, skin, etc, comprising injecting, flowing, pumping,
inhaling, transfusing or by other means exposing a sample behind a
barrier, and detecting said misfolded target protein behind or inside
said barrier by appropriate means of detection. Means for detection and
administration of CPEs may vary depending on side-chain or end-terminal
functionalization according to the present invention and are exemplified
elsewhere in the description.

[0038]Another aspect of the invention is a bioassay which relies on the
complete distinction during the capture step of amyloid, misfolded or
pathological forms of proteins in the presence of native proteins using
CPE and later detected by appropriate means, such as colorimetric,
absorbance or fluorescence based methods. It is very important to avoid
false positives but also to avoid false negatives. Selective proteolysis
of native proteins can be, but should not be required, a way to achieve
this.

[0039]A still further aspect of the invention relates to the uniqueness of
CPEs providing not only capture of misassembled, misfolded, aggregated or
pathogenic forms of proteins, but also being capable of acting as a
capture agent and at the same time report said capturing event, if
desired, and be translated into optical and detectable signals.

[0040]The multiplicity of misfolded proteins that one may wish to identify
also implies that the invention in a still further aspect can be
implemented in the form of a microarray, and which calls for anchoring
and patterning of the detecting system on a surface. The conjugated
polyelectrolytes of the present invention are then used as capture agents
of misfolded proteins, either in solution or on a solid support, and, if
desired or required, used as a detection agent for said capture event.

[0041]The full scope of the invention is that defined by the appended
claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1: The chemical structure of some selected examples of
different CPEs based on poly-thiophene backbone and mixed
poly-thiophene-benzene backbone. Side chain variations and defined
backbone sizes can be constructed. These have been given a name for
simplicity POWT, PTAA, POMT, PT2, tPOWT, tPTM1, f-PONT, L-POMT and
D-POMT.

[0043]FIG. 2: Functionalized CPEs. R1 and R2=Functionalized at the end
terminal/terminals. R1 and R2 can be the same or different. Rs=side
chain.

[0044]FIG. 3: Schematic capture of an arbitrary amyloid polymer using
either an end-terminal functionalized CPE or one without the end-terminal
functionalized. The drawing is not drawn to scale and does not represent
the size relationship between the CPE and the amyloid. The number of
amyloid monomers in a misfolded protein can be between one and many in
the present invention. Also, the CPEs can be immobilized on a solid
support and examples of this are shown in later figures. The number of
CPE molecules, functionalized or not, can be anything from one to many.
Side chains (Rs) are not shown in the schematic.

[0045]FIG. 4: Schematic drawing showing capture of misfolded protein by
using a CPE immobilized on a solid support. The sample is exposed to the
CPE, being functionalized or not, localized on a solid support whereby
the CPE captures the misfolded proteins in said sample. The drawing is
not drawn to scale and does not represent the size relationship between
the CPE and the misfolded protein. The number of CPE molecules,
functionalized or not, can be anything from one to many. Side chains (Rs)
are not shown in the schematic.

[0046]FIG. 5: Schematic drawing showing capture of misfolded protein on a
suitable solid support, such as glass slide, glass bead, filter matrix,
separation matrix, etc, using a CPE. The sample is exposed to the CPE,
being functionalized or not, localized on a solid support whereby the CPE
captures the misfolded proteins in said sample. The drawing is not drawn
to scale and does not represent the size relationship between the CPE and
the misfolded protein. The number of CPE molecules, functionalized or
not, can be anything from one to many. Side chains (Rs) are not shown in
the schematic.

[0047]FIG. 6: Principle of detecting PrP-amyloid stained by the CPE PTAA
using gravimetric sedimentation. The size of the crystal structure of PrP
is also given as a size reference to the amount of PrP in the microscopic
aggregates.

[0048]FIG. 7: Schematic drawing showing capture of misfolded protein using
a magnetic bead coated with a CPE. A magnetic bead, for example coated
with avidin, is coated with a functionalized CPE, for example
functionalized with biotin. The sample is exposed to the magnetic bead
with the CPE whereby the CPE captures the misfolded proteins in said
sample. The drawing is not drawn to scale and does not represent the size
relationship between the CPE and the misfolded protein. A magnet can be
used as a collection/concentration step. The number of CPE molecules can
be anything from one to many. Side chains (Rs) is not shown in the
schematic. An optional detection step can be included after the capture
and the collection/concentration step using a magnet.

[0049]FIG. 8: Schematic drawing showing capture of misfolded protein using
a biotin functionalized CPE and an avidin coated surface. The
functionalized CPE can be as is or on a bead. The sample is exposed to
the CPE whereby the CPE captures the misfolded proteins in said sample. A
avidin coated surface can be used as a collection/concentration step. The
drawing is not drawn to scale and does not represent the size
relationship between the CPE and the misfolded protein. The number of CPE
molecules can be anything from one to many. Side chains (Rs) is not shown
in the schematic. An optional detection step can be included after the
capture and the surface collection/concentration step.

[0050]FIG. 9. Fluorescence spectra of 0.5 mg/ml PTAA with insulin in a
phosphate buffer 20 mM pH 8, before and after filtration through 0.22
μm filter.

[0053]FIG. 12: Capture of fibrils using centrifugation of PTAA-fibril
complexes, followed by fluorescence measurements.

[0054]FIG. 13: Fluorescence spectra demonstrating the interaction between
a few selected CPEs and Gd2O3 nanoparticles. The measurement
was performed in double distilled water.

DETAILED DESCRIPTION OF THE INVENTION

[0055]In general terms, the present invention relates to novel methods for
capturing misassembled, misfolded or aggregated forms of proteins,
especially amyloid fibrils and pathogenic forms, using conjugated
polyelectrolytes. The misfolded protein is exposed to the conjugated
polyelectrolyte (CPE) whereby the CPE captures, binds or isolates said
misfolded protein of interest. The capture can be performed with high
affinity or high selectivity for the misfolded protein compared to native
folded proteins. The capture is performed under conditions in which
conjugated polyelectrolytes bind misfolded altered proteins, like in
PrPSc or A-beta, and preferably, but not necessary, where such conjugated
polyelectrolytes bind these abnormal forms but do not bind their
non-aggregated normal native forms. It is desired that the non-covalent
binding between the CPE and misfolded protein of interest occurs with
sufficiently high affinity and under chosen conditions and sufficiently
selective to be useful in assays were the aggregated, misfolded altered
protein can be captured or isolated from a sample.

[0056]According to the present invention the terms capture, binding or
isolation are subsets of the general term which is separation. Separation
of misfolded proteins in a sample, for example to remove them from said
sample, can be achieved using CPEs and has never been described before.
For example, a sample solution can be flowed over or inside a solid
support having a CPE/CPEs immobilized onto said solid support. The action
of said CPE/CPEs is to capture the misfolded protein from the sample, but
at the same time the CPE/CPEs also separates the misfolded protein from
the sample.

[0057]The term "capture" as used in this application means a probe that
can bind a misfolded protein, ex vivo or in vivo, and thereby separate,
isolate or localize it in the presence of the non-aggregating normal form
of the protein.

[0058]The invention is based on non-covalent capturing of misfolded,
abnormal, misassembled, aggregating or pathogenic proteins using a
conjugated polyelectrolyte interacting with said misfolded protein. The
interaction occurs without covalent bonding and is based on dipole-dipole
bonding, hydrogen bonding, electrostatic- and non-polar interactions
between the conjugated polyelectrolyte and the misfolded protein, herein
referred to as non-covalent bonding, which further includes any type of
bonding that is not covalent in nature.

[0059]Some aspects of the present invention might provide for covalent
attachment of conjugated polyelectrolytes to some entity, such as
proteins, misfolded proteins, peptides, biomolecules, other molecules or
surfaces.

[0060]One aspect of the invention is thus a process where conjugated
polyelectrolytes is used for separation, for example the selective
capture, binding or isolation of misfolded protein, an aggregating
abnormal form or a pathological form of a protein in the presence of the
native form of the protein. A sample is exposed to a conjugated
polyelectrolyte, either localized on a solid support, on a bead of any
size, as a hydrogel or provided as solution, whereby the conjugated
polyelectrolyte captures, binds or isolates any misfolded, aggregated,
misassembled or pathogenic protein. The method can be performed under
such conditions that the high avidity conjugated polyelectrolyte can
selectively capture said misfolded protein with high selectivity for said
misfolded form in the sample containing both native and normal forms of
proteins as well as the misfolded forms.

[0061]The conjugated polyelectrolyte may be immobilized on a solid support
of suitable kind and applicable shape, where the surface of the solid
support presents the conjugated polyelectrolyte to the sample. The shape
and size of the solid support may be of any kind, for example circular,
flat or a bead, and surface may be modified with conjugated
polyelectrolytes by non-covalent means or covalently bonded within the
structure of the solid support or attached to the surface of said solid
support. A solid support according to the present invention also
constitutes tubes, dialysis equipment, pumps, insulin pumps, equipment
for administrating proteins, syringes and needles. When localized on a
solid support the conjugated polyelectrolyte can be in any suitable form,
for example as a hydrogel, as polymer film, free standing CPEs,
scaffolded or layered. The CPEs can also be provided free in solution
when used as an agent to capture misfolded proteins. If the CPE is
provided free in solution any means to remove the misfolded protein after
being captured by the CPE can be applied, for example sedimentation,
centrifugation, adsorption, absorption, drying, boiling or evaporation.
Optionally the CPE can be functionalized to provide for means of
detection, immobilization, facilitate capture, enhanced selectivity,
enhanced affinity or other things of significance for the assay.

[0062]The described capturing, binding or isolation conditions may include
the presence of a competition agent provided to the sample, which
competition agent has a lesser binding avidity for the misfolded form of
the protein than the conjugated polyelectrolyte. Generally, if a
competition agent is used it has lesser affinity or selectivity than the
conjugated polyelectrolyte.

[0063]Optionally, the surface of the solid support can be coated thereon
or bonded thereto with the conjugated polyelectrolytes.

[0064]It has been reported in the scientific literature that amyloid forms
of proteins, or at least protease sensitive forms of misfolded proteins,
can interact with polyanions. These negatively charged polymers might be
interacting with a positively charged region of the misfolded protein
structure and there could be multiple interactions with the aggregated
proteins. We find that a conjugated polyelectrolyte is able to bind to
the misfolded protein aggregate or smaller associations of misfolded
proteins, which might be even as few as two monomers of the amyloid
protein, structure with much higher avidity than these polyanions, and it
might therefore even displace the polyanion compounds. One possible
mechanism for the high avidity of CPEs is that CPEs can form both
polyionic as well as hydrophobic interactions with the misfolded protein
with its charged substituents and hydrophobic conjugated backbone at the
same time. Thus, conjugated polyelectrolytes immobilized to a surface or
in solution can capture specifically the abnormal protein. Native
proteins are non-aggregating and do not generally have such a high
affinity interaction with polyanions. The assay conditions can be chosen
such that the presence of lower affinity anions such as the detergent
Sarkosyl improves capture specificity still further by competing with the
immobilized CPE or a CPE in solution.

[0065]Separation, for example capture, binding or isolation, of a
misfolded protein, an aggregating abnormal form or a pathological form of
a protein in a sample using a CPE can be coupled to a change of a
property of said CPE in response to the capture, binding or isolation of
said misfolded protein. The changed property of the CPE can be detected
by for example optical means, like fluorescence or absorption, which are
then used to determine when said capturing event has occurred.

Other Methods of Detection

[0066]Said conjugated polyelectrolytes can be functionalized according to
aspects of the present invention to enable efficient or desired way of
detecting the separation. Other methods of detecting the conjugated
polyelectrolyte, that is bonded to or is in contact with said misfolded
protein, is done by the appropriate method, such as two- or multiphoton
spectroscopy, magnetic resonance image (MRI), PET, etc. One example to
image the interaction between the CPE and the misfolded protein behind a
barrier, such as a tube, a membrane, a tube, skin, etc, is described
here, but there are other obvious ways. A conjugated polyelectrolyte or a
functionalized conjugated polyelectrolyte can be detected by some means
behind said barrier using for example two- or multiphoton spectroscopy or
magnetic resonance image (MRI), is then used to determine when said
separation, for example capturing or binding event, has occurred.

CPEs as Therapeutic Agents

[0067]The invention also relates to the use of conjugated polyelectrolytes
as novel therapeutics where one way of action is by interfering with
formation of amyloid protein in vivo. The CPEs can alter amyloid
pathology upon injection into organism, i.e. influence disease
pathogenesis by acting as therapeutic pharmacophores. Therapeutic agents
based on CPEs for protein misfolding diseases, and in particular
Alzheimer's and prion diseases where an ante-mortem diagnosis and therapy
would have a huge impact and a great benefit for public health, is
accordingly envisioned in the present invention.

[0068]In the one embodiment present invention provides a method where a
conjugated polyelectrolyte directly captures said misfolded protein. The
capture in this case occurs without covalent bonding and is based on
dipole-dipole bonding, hydrogen bonding, electrostatic- and non-polar
interactions between the conjugated polyelectrolyte and the biomolecule,
herein referred to as non-covalent bonding, which further includes any
type of bonding that is not covalent in nature.

[0069]The term "direct capture" as used in this application means a probe
that can directly capture a misfolded protein by direct binding to said
misfolded protein and thereby isolate or localize it in a sample, without
the need for other macromolecular compounds.

[0070]The term "avidity" as used in this application shall be taken to
mean, as usual, the overall binding strength of a molecule with many
binding sites with a multivalent binding agent. This should been seen in
contrast to "affinity", being the binding strength between each
individual binding site and of the molecule and the binding agent.

[0071]It is possible to suitable implement the conjugated polyelectrolyte,
either functionalized or not, as an active part of a capture device, e.g.
by immobilizing the conjugated polyelectrolyte on a substrate. The
capture device comprises a suitable receptacle for said substrate, and a
complex between the conjugated polyelectrolyte and the misfolded protein
is formed on the substrate. Optionally, said CPE is used to detect the
capturing event at the same time as it occurs.

[0072]However, other configurations are possible, e.g. the conjugated
polyelectrolyte can be provided in solution and passed through a flow
cell while a protein solution is mixed with the flow of complex solution.
The capture can be monitored by various analytical techniques.

[0073]In particular the present invention allows capturing of misfolded
proteins, associated with diseases, using a conjugated polyelectrolyte
interacting with the misfolded protein. Capture of misfolded proteins is
essential in the development of methods for diagnosis of diseases related
to misfolded proteins, to remove a misfolded protein from a sample,
detecting the separation of misfolded proteins in a sample behind a
barrier as well as various therapeutic techniques based on conjugated
polyelectrolytes.

[0075]The functional groups added to the side chain (Rs) or on the
end-terminals (R1 and R2), are shown in FIG. 2. These can be, but are not
limited to, charged anionic, cationic or zwitterionic with pKa at
different pH, this makes these polythiophene derivatives suitable for
forming polyelectrolyte complexes with negatively or positively charged
oligomers and polymers. In addition, the ionic groups create versatile
hydrogen bonding patterns with different molecules. One of the proposed
reasons, the invention shall not be bound to this proposal, for CPEs
unique ability for capturing misfolded proteins is the avidity. One way
to look at the misfolded proteins can be seen as a polyionic molecule
having an alternative or repetitive charge distribution along the axis of
the molecule. The tailor made CPEs of the present invention can form
strong non-covalent interactions with the misfolded proteins and
therefore capture them, see the schematic illustration in FIG. 3. The
end-terminal functionalized conjugated polyelectrolytes where the
functional group can be, but is not limited to, DNA, RNA, peptides, amino
acids, histidine, histidine10, proteins, peptide scaffolds, biotin,
avidin, streptavidin, chelators, active groups, antibodies, affinity
peptide scaffolds, scFV, FAB, enzymes, ligands, receptor ligands,
steroids, biomolecules or other molecules, nanoparticles, microparticles,
gold nanoparticles, gold microparticles, magnetic beads, protein coated
beads, peptide coated beads, supramagnetic beads or particles, gadolinium
nanoparticle, gadolinium oxide particles, Gadolinium ions, lanthanide
doped nanoparticles, lanthanide-doped gadolinium oxide nanoparticles,
lanthanide particles, can provide for surface immobilization, secondary
capture or means for detection behind a barrier according to the present
invention.

[0076]The CPE may be selected to have the ability under non-selective
conditions to capture both misfolded, aggregating altered or rogue forms
of a protein and also the non-aggregating normal form of the protein as
well as the ability to capture the misfolded protein selectively under
selective conditions. The CPE, either localized on a solid support or in
solution, can, if desired, under both non-selective conditions and
selective conditions change its optical properties in such way that it is
possible to distinguish if the CPE has captured a misfolded or native
protein. The appropriate non-selective conditions or selective conditions
assay conditions may be achieved by adjusting the sample, reaction or
washing solution in terms of pH, ionic strength, choice of buffer or by
adding salts, ions, metal ions, detergents, polyelectrolytes, proteins,
particles or chelators.

[0077]It is furthermore possible to add one or more agents to the sample,
reaction or washing solution capable of increasing differentiation of CPE
binding the misfolded protein from CPE interacting with normal protein
and in such way increase the selectivity of capturing the misfolded
protein in a given sample. The CPE in this case may be provided as
localized on a solid support or in solution. Such agents include
detergents, such as Triton-X, Saponin, SDS, Sarkosyl,
n-laurosylsarcosine, fatty acid sarcosines, CHAPS, Brij,
Octyl-b-glycoside, Tween 20, Nonidet P-40 or other variants, ions and
salts, such as metal ions, molecular ions and organic ions, chelators,
such as EDTA, EGTA, 2,2'-Bipyridyl, Dimercaptopropanol, lonophores,
Nitrilotriacetic acid, ortho-Phenanthroline, Salicylic acid and
Triethanolamine, solvents, such as water, alcohols, organic solvents,
chlorinated solvents, aminated solvents and sulfonated solvents, and
other agents, such as polymeric materials, polyelectrolytytes
(zwitterionic, anionic or cationic), Carbohydrates (including
polysaccharides), Organic acids with more than one coordination group,
Lipids Steroids, Amino acids and related compounds, Peptides, Phosphates,
Nucleotides, Tetrapyrrols, Ferrioxamines, lonophores, such as gramicidin,
monensin and valinomycin Phenolics. Other agents include proteins, such
as Trypsin, Proteinase K, antibodies, serum albumine and others.

[0078]The conjugated polyelectrolyte can be suitably implemented as an
active part of a device that capture, isolate, bind or remove misfolded
proteins in a sample. This can be achieved by, for example, immobilizing
the conjugated polyelectrolyte on a substrate which then is exposed to a
sample by appropriate means (see FIGS. 4 and 5 for two examples).
Suitably the device comprises a suitable receptacle for said substrate,
and an interaction between the conjugated polyelectrolyte and the
misfolded protein is formed on the substrate whereby it captures,
isolates, binds or removes misfolded proteins in the sample. The
conjugated polyelectrolyte can be a part of a system using an antibody
that recognizes or identifies the misfolded protein (for example
PrPSC, PrPres, amyloid-beta, fibrils formed of Aβ(1-40),
Aβ(1-42) or Aβ(1-43), amyloid-β (Aβ) peptides,
beta-2-microglobulin molecules in dialysis-related amyloidosis (DRA),
IAPP, alpha-synuclein in Parkinson's disease or huntingtin in
Huntington's disease) that has been or will be captured by the CPE.
Optionally, the conjugated polyelectrolyte can be used as an optical
probe to report the capture of the misfolded protein (see FIGS. 4 and 5
for two examples). However, other configurations are possible, e.g. the
conjugated polyelectrolyte can be provided in solution for capturing, and
at the same time staining, a misfolded protein in a sample or in vivo. An
antibody, in solution or on a solid support, can then be used identify or
recognize the misfolded protein.

[0079]The detailed description of the invention that follows will deal
separately with the conjugated polyelectrolytes, misfolded, abnormal,
aggregating, rouge or pathogenic protein, diseases related to said
misfolded proteins, methods of capturing said misfolded proteins from a
sample solution, immobilization of conjugated polyelectrolytes onto solid
supports, and arrays. The invention is finally exemplified with a number
of experiments demonstrating the utility thereof.

I Conjugated Polyelectrolytes

[0080]The present invention relates to a variety of conjugated
polyelectrolytes, with a minimum of 3 mers, consisting of mers derived
from the monomers thiophene, pyrrole, aniline, furan, phenylene,
vinylene, fluorene, ethylenedioxythiophene or their substituted forms,
forming homopolymers and copolymers thereof. The conjugated
polyelectrolyte can be mono dispersed, consist of polyelectrolyte chains
with a well-define chain length, or poly dispersed, comprise of
polyelectrolyte chains with different chain length. Furthermore, monomers
with anionic-, cationic or zwitterionic side chain functionalities are
included within the scope of the invention. The side chain
functionalities is derived from, but not limited to, amino acids, amino
acid derivatives, neurotransmittors, monosaccharides, nucleic acids, or
combinations and chemically modified, L and D enantiomers, or derivatives
thereof. The conjugated polyelectrolytes of the present invention may
contain a single side chain functionality or may comprise two or more
different side chain functionalities. The functional groups of the
conjugated polyelectrolytes, charged anionic or cationic at different
pHs, make these polyelectrolyte derivatives suitable for forming strong
polyelectrolyte complexes with negatively or positively charged oligomers
and polymers. In addition, the ionic groups create versatile hydrogen
bonding patterns with different molecules.

[0081]Some aspects of the present invention might provide for covalent
attachment of conjugated polyelectrolytes to some entity, such as
proteins, misfolded proteins, peptides, biomolecules or other molecules.

[0082]The functional groups added to the side chain (Rs) or on the
end-terminals (R1 and R2), in FIG. 1 or FIG. 2, can be, but not limited
to, charged anionic, cationic or zwitterionic with pKa at different pH,
this make these polythiophene derivatives suitable for forming
polyelectrolyte complexes with negatively or positively charged oligomers
and polymers. In addition, the ionic groups create versatile hydrogen
bonding patterns with different molecules. One of the proposed reasons,
the invention shall not be bound to this proposal, for CPEs unique
ability for capturing misfolded proteins is the avidity. One way to look
at the misfolded proteins can be seen as a polyionic molecule having an
alternative or repetive charge distribution along the axis of the
molecule. The tailor made CPEs of the present invention can form strong
non-covalent interactions with the misfolded proteins and therefore
capture them, see the schematic in FIG. 3. The end-terminal
functionalized conjugated polyelectrolytes where the functional group can
be, but not limited to, DNA, RNA, peptides, amino acids, histidine,
histidine10, proteins, peptide scaffolds, biotin, avidin,
streptavidin, chelators, active groups, antibodies, affinity peptide
scaffolds, scFV, FAB, enzymes, ligands, receptor ligands, steroids,
biomolecules or other molecules, nanoparticles, microparticles, gold
nanoparticles, gold microparticles, magnetic beads, protein coated beads,
peptide coated beads, supramagnetic beads or particles, gadolinium
nanoparticle, gadolinium oxide particles, Gadolinium ions, lanthanide
doped nanoparticles, lanthanide-doped gadolinium oxide nanoparticles,
lanthanide particles, can provide for surface immobilization, secondary
capture or means for detection behind a barrier according to the present
invention.

II Misfolded Protein

[0083]The conjugated polyelectrolytes of the present invention capture a
misfolded protein of interest. In the present invention the term
misfolded protein should be taken it its broadest sense, including
misassembled proteins, pathological or rogue forms of proteins, i.e.
amyloid form, misfolded form or aggregated forms of protein, as plaques,
preferably the misfolded proteins can be amyloids which are insoluble
fibrous protein aggregations sharing specific structural traits.

[0084]The capture occurs without covalent bonding and is based on
dipole-dipole bonding, hydrogen bonding, electrostatic- and non-polar
interactions between the conjugated polyelectrolytes and the misfolded
protein. The conjugated polyelectrolyte might interact with both a normal
native protein and the misfolded protein. The conjugated polyelectrolytes
can also capture amyloid or misfolded proteins prepared in vitro. Without
being bound by theory, it is the present hypothesis that the conjugated
polyelectrolyte can capture misfolded specifically and with high affinity
under suitable assay conditions.

[0085]The misfolded protein can be chemically modified to be captured by
the conjugated polyelectrolyte of choice. Methods of derivatizing a
diverse range of proteins are well known. For example, amino acid side
chains can easily be modified to contain polar and non-polar groups or
groups with hydrogen bonding abilities. The misfolded protein can be in
solution, on a solid phase, in vivo, in vitro or in tissue samples and
the capture of misfolded proteins can be made in water solutions, organic
solvents, body fluids, on a solid phase or in tissue samples.

[0086]Samples, either in vivo or in vitro, of interest, that may contain
misfolded proteins, include, but is not limited to, blood, brain,
peripheral tissue, homogenized brain, homogenized peripheral tissue,
fluids, biopsies, urine, cerebrospinal fluid (CSF), lymph and other
samples of relevance. Assays based on capture of misfolded proteins are
of great commercial interest, of huge diagnostic value, as therapeutic
agents as well as selective assays for capture of misfolded proteins in
blood (screening of blood products) or other complex media. Staining of
tissue sections, from brain, muscle, fat, mucus, nerve, blood vessels or
other tissues.

III Diseases Related to Misfolded Proteins

[0087]Diseases featuring amyloid proteins are relevant examples for the
description of diseases related with misfolded proteins, where
amyloidosis are known as a disease and may be inherited or acquired. Note
that amyloidosis by default usually refers to AA amyloidosis, but any
disease related to amyloid proteins, which presents amyloid deposition,
is an amyloidosis. For example CJD, vCJD, Alzheimer's and diabetes are
almost never referred to as amyloidoses.

[0088]In this paragraph some examples of amyloidosis with relevance to the
present is named. Primary amyloidosis includes mutations in lysozyme,
transthyretin, apolipoprotein B, fibrinogen and AL amyloidosis
(immunoglobulin light chains, as seen with multiple myeloma). Secondary
amyloidosis includes AA amyloidosis (serum amyloid A protein, an
acute-phase protein due to chronic inflammation) and Gelsolin amyloidosis
(plasma gelsolin fragments). Familial or hereditary amyloidosis, are most
commonly caused by mutations in the transthyretin protein, but in rare
occurrences can also be caused by apolipoprotein A1, gelsolin,
fibrinogen, and lysozyme mutations, primarily caused by genetics,
believed to be autosomal dominant, high probability of passage to
offspring, Appalachian type amyloidosis and Shar Pei fever for
amyloidosis in Shar Peis. Examples of organ-specific amyloidosis are
Diabetes mellitus type 2 (amylin, also known as IAPP), Neurology,
Alzheimer's disease (Aβ 39-42), Parkinson's disease
(alpha-synuclein), Huntington's disease (huntingtin), Transmissible
spongiform encephalopathies (prion protein, PrP), some examples are
Creutzfeldt-Jakob disease (PrP in cerebrum), Kuru (diffuse PrP deposits
in brain), Fatal Familial Insomnia (PrP in thalamus) and Bovine
spongiform encephalopathy (PrP in cerebrum of cows), Congophilic
angiopathy (Amyloid beta). Cardiac amyloidosis includes congestive heart
failure; some instances (PrP or transthyretin in heart). Inclusion body
myositis. Another important example are the Iatrogenic conditions like
insulin amyloidosis, believed to be caused by injection-administered
insulin.

[0090]The prion diseases [e.g. bovine spongiform encephalopathy (BSE), and
Creutzfeldt-Jakob disease (CJD)], are associated with the conformational
conversion of the normal cellular prion protein, (PrPC), to an
infectious disease-associated isoform denoted PrPSc. The misfolded
infectious form of the protein, PrPsc is the cause of a group of
rare, fatal brain diseases, called prion diseases that affect humans and
mammals. The prion diseases are also known as transmissible spongiform
encephalopathies (TSE), and they include bovine spongiform encephalopathy
(BSE, or "mad cow" disease) in cattle; scrapie in sheep; chronic wasting
disease in deer and elk; and in humans [Creutzfeldt Jakob disease (CJD),
Gerstmann-Straussler-Scheinker disease (GSS), Kuru]. The conjugated
polyelectrolytes of the present invention are intended to be used for
methods for capture of pathogenic prions associated with these diseases.

IV Methods of Detection

[0091]As already indicated the present invention is based on the
utilization of conjugated polyelectrolytes, functionalized conjugated
polyelectrolytes or combinations thereof to capture, bind, isolate or
remove a misfolded protein from a sample. The capture event can be
observed for example by, but not limited to fluorescence, Forster
resonance energy transfer (FRET), quenching of emitted light, absorption,
two- or multiphoton spectroscopy, magnetic resonance image (MRI), dynamic
light scattering (DLS), an electrical or magnetic response,
electrochemistry, amperometry, coulometry, gravimetry, polarization or
anisotropy, fluorescence correlation spectroscopy (FCS), quartz crystal
microbalance, quartz crystal microbalance with dissipation or other
techniques and physical properties. The emission intensities can be
recorded by a fluorometer and enhancement of the photon flow in the
detector can increase the sensitivity. This can be achieved using a lamp
or a laser as the excitation source. The fluorometric change can also be
detected by the use of a fluorescence microscope or a confocal
microscope. A combination of excitation or emission filter can be used
and the picture can be recorded by a CCD-camera, video camera, regular
camera or by a Polaroid camera. The pictures can then be analyzed by
image processing software on a computer, Image correlation spectroscopy
(ICS) or by other means.

[0093]Immobilization of the conjugated polyelectrolytes is achieved by
physical adhesion or covalent attachment to the solid support, and can be
performed at elevated temperatures or by entrapment in a hydrogel matrix.
Immobilization of the conjugated polyelectrolytes of the present
invention may be desired to improve their ease of use, assembly into
devices (e.g. arrays), stability, robustness, fluorescent response, to
fit into the process of high-throughput-screening (HTS) using micro titer
plates and other desired formats.

[0094]Solvents for the conjugated polyelectrolytes of the present
invention and the prion proteins during the immobilization to the solid
support can be, but are not limited to, water, buffered water solutions,
methanol, ethanol and combinations thereof. Supporting polymers of other
kinds can also be added in this step.

[0095]Biomolecules, misfolded proteins, antibodies, proteins, peptides,
misfolded proteins or other molecules can also be immobilized on a solid
support or in microtiter wells. A recognizing, identifying or capture
antibody, or other biomolecules with selective capabilities, can also be
immobilized, either by its own, together with the conjugated
polyelectrolyte (i.e. mixed with the polyelectrolyte solution) or later
recognizing the CPE that has captured a misfolded protein. When the
biomolecules, misfolded proteins, antibodies, proteins or peptides are
immobilized on the solid support together with the conjugated
polyelectrolyte of the present invention they form a complex with the
polyelectrolyte through non-covalent interactions. This complex is formed
without covalent chemistry and is based on hydrogen bonding,
electrostatic- and non-polar interactions between the conjugated
polyelectrolyte and the prion protein.

VI Arrays and Lines

[0096]According to the present invention the generation of large arrays of
the same or different conjugated polyelectrolytes in each spot or line
can overcome shortcomings of a single sensor or a solution based
approach. The array or parallel line approach opens up the parallel
analysis of one or different samples that may contain misfolded proteins
to one or different-conjugated polyelectrolytes in an easy way, where at
least one step in the protocol utilizes a CPE to capture the misfolded
protein. The main purpose of using arrays is to increase ease of use,
portability, quantification, selectivity among other qualities and
characteristics. With this approach we can explore the ability to measure
multicomponent samples and to use partially selective sensor spots. This
gives the opportunity to analyze two or more samples of interest at the
same time and to do on-chip determinations. By immobilizing the
conjugated polyelectrolyte and/or biomolecules, misfolded proteins, prion
protein, antibodies, proteins or peptides on solid supports of any size
and in any chosen patterns (such as arrays, lines, spots, posts) small,
portable, easily read and interpretable devices can be constructed.

[0097]The use of multiple arrays requires that detection can be done for a
great number of samples, more or less simultaneously. This is often done
in the form of a microarray, where many individual detector elements (or
probes) are integrated on a small surface area, to allow for massive
parallelism in the detection. We have shown that the conjugated
polyelectrolyte and the conjugated polyelectrolyte/protein complexes can
be printed by micro contact printing using elastomer stamps. Transfer
onto a microarray surface may also be done by spotting conjugated
polyelectrolyte solutions, or by ink jetting polyelectrolyte solutions or
by the other methods mentioned above. These steps are essential to
prepare a multipixel microarray.

EXAMPLES

Example 1

Capture and Detection of PrP-Amyloid in Solution Using Conjugated
Polyelectrolytes

[0098]Surface detection by means of fluorescence microscopy PrP and
PrP-amyloid sample, the latter being PrPSc- or PrPres-like, is
contained in a test tube. PrP-amyloid was generated through dialysis of 1
mg/ml recHuPrP90-231 dissolved in 3 M guanidinium hydrochloride versus
water. Another protocol to generate PrP-amyloid is by shaking in a test
tube at a high salt concentration. Adding the conjugated polyelectrolyte
PTAA (10 μg/ml) to this solution, containing PrP and PrP-amyloid,
causes PTAA to capture, bind and stain as well, PrP-amyloid. The
PTAA/PrP-amyloid formation can then be left to sediment to the bottom of
the test tube, and can then easily be isolated from the sample and
detected if desired. Other ways to collect PTAA/PrP-amyloid is, but not
limited to, filtration, centrifugation, capture on a hydrophobic surface,
capture on a surface with covalently attached PTAA, using functionalized
particles, salt precipitation and by immunoprecipitation. Methods for
detection or visualization of PTAA/PrP-amyloid includes, but not limited
to, fluorescence microscopy, fluorescence detection in plate readers or
spectrofluorometers, absorption detection in plate readers or
spectrometers, array fluorescence reader, photodiodes, fluorescence
polarization or anisotropy, circular dichroism and more. This method also
provides a way to capture, clean, remove or filter PrP-amyloid,
PrPSc, PrPres from a given sample in solution. In this
particular example PTAA/PrP-amyloid formation was collected on a glass
substrate and visualized using a fluorescence microscope, see FIG. 6.
Using this instrument the resolution allows identification of individual
PrP-amyloid particles smaller than 1 μm×1 μm×1 μm
which corresponds to less than 2 pg of PrP.

Example 2

Filtration, Capture

[0099]In order to separate native and fibril insulin and hence purifying
the fibril insulin, filtration of polymer-insulin samples were performed.
Samples tested were native insulin and fibril insulin, with and without
PTAA. Initial PTAA concentration were 0.5 mg/ml and insulin concentration
2 mg/ml, and samples contained 10 μl PTAA+25 μl insulin to 1 ml in
phosphate buffer 20 mM pH 8. For all samples fluorescence measurements
were performed both before and after filtration, and the filters were
opened and visually inspected. Filters used were 0.22 μm Millex-gu,
and 1 ml of every sample were filtered through the filters.

[0100]The fluorescence spectra of PTAA-native insulin (PTAA-ins) and
PTAA-fibril insulin (PTAA-fib) are shown in FIG. 9 before and after
filtration through the 0.22 μm filter. Before filtration the spectra
look as expected. After filtration it is obvious that samples with PTAA
and fibril insulin is retained in the filter (seen as a decrease in
fluorescence), while samples with native insulin does not affect the
passing of the polymer. Thus, filtration could be used to remove native
insulin bound to PTAA. A visual inspection of the filters after
filtration show that aggregates of PTAA-fibril insulin is present on the
filters (seen as red aggregates), while filters with PTAA-native insulin
show no aggregates or change in colour.

[0101]Measurements to detect the presence of PTAA-fibril insulin complexes
caught in the filter were also done. Samples with PTAA alone and with
native insulin containing 0, 5, 50 and 100% fibril insulin were prepared
as described above. The content of native insulin were decreases as the
content of fibril insulin increased, resulting in the same total insulin
content in all samples. The samples were filtrated through a 0.8 μm
Millex-aa filter and fluorescence was measured on the filtrate. The
filters were then back-filtered, by pressing 1 ml buffer through the
filters backwards. Fluorescence was then measured on the back-filtered
filtrate.

[0102]The fluorescence spectra of the first filtrate is shown in FIG. 10.
Increasing fibril content leads to more PTAA-fibril insulin complexes
being retained in the filter, while PTAA can pass freely with native
insulin. The back-filtered samples are shown in FIG. 11. While most
complexes are still caught in the filters, it is obvious that increasing
fibril content leads to more PTAA-fibril insulin complexes being resolved
into solution following back-filtration. Thus PTAA can be used to capture
fibril insulin.

[0103]Using filtration to capture PTAA-fibril complexes is not limited to
the filters and examples above. Any other type of filter, matrix,
buffers, concentrations, and additives can be used, and analysis and
detection can be obtained by fluorescence measurements, absorbance
measurements, visual inspection, microscopic methods, fluorescence
polarization or anisotropy, circular dichroism and more.

Example 3

Capture and Detection of Insulin-Amyloid in Solution Using Conjugated
polyelectrolytes. Solution Detection by Means of Fluorescence
Measurements.

[0104]Samples with native insulin containing 0, 1, 5, 50 and 100% fibrils
(2 mg/ml, 25 μl) are mixed with PTAA (0.05 mg/ml, 10 μA) and 965
μA 20 mM phosphate buffer pH 7.0 in a test tube. Fibril insulin was
generated through incubation of native insulin at 65 degrees Celsius at
pH 2 for 8 hours. The samples were centrifuged at 10 000 rpm for 5
minutes, the supernatant was removed, new buffer was added, and the
procedure was repeated once. The PTAA binds to the fibril insulin and
sediments to the bottom of the test tube under the centrifugation. The
samples were then measured using a fluorescence microplate reader.
Reference samples of PTAA-native insulin and PTAA-fibril insulin that had
not been centrifuged were also measured. The results are shown in FIG.
12.

[0105]The PTAA-fibril aggregates sediments to the bottom of the test tube
and can easily be collected for detection by removing the supernatant
following centrifugation. By resolving the pellet the PTAA-fibril
complexes can then be detected by fluorescence measurements. Methods for
detection or visualization of PTAA/PrP-amyloid includes, but not limited
to, fluorescence microscopy, fluorescence detection in plate readers or
spectrofluorometers, absorption detection in plate readers or
spectrometers, array fluorescence reader, photodiodes, fluorescence
polarization or anisotropy, circular dichroism and more. This method also
provides a way to capture, clean, remove or filter amyloids and fibrils
from a given sample in solution. As can be seen in this example,
increasing the amount of fibrils leads to more PTAA-fibril complexes.
From 1% fibrils up to 100% fibrils the fluorescence intensity increases,
showing the binding between PTAA and the fibrils, as no PTAA at all is
present following centrifugation with native insulin.

Example 4

Immobilization of CPEs to Solid Supports

[0106]This example describes the immobilization of PTAA to a glass surface
that has been coated with APTES. The silane APTES was in order to have
amine groups for the chemical coupling. EDC (or EDAC) is a zero-length
crosslinking agent used to couple carboxyl groups to primary amines. This
crosslinker can be used in diverse applications such as forming amide
bonds in peptide synthesis, attaching haptens to carrier proteins to form
immunogens, labeling nucleic acids through 5' phosphate groups and
creating amine-reactive NHS-esters of biomolecules.

[0107]The CPEs having a carboxyl group, --COOH, (such as PTAA, POWT, tPTAA
or tPOWT) can be activated first and then coupled to an amine surface of
any kind, such as functionalized magnetic beads. CPEs that does not
contain any carboxyl groups but have amine groups (such as POMT, tPOMT
and PTT) can be coupled to an activated carboxyl surface. If you have a
magnetic bead with carboxyl groups just put this one into the EDC/NHS
solution instead.

Protocol, Immobilization of PTAA

[0108]Clean a glass slide using TL1 wash (5 parts H2O, 1 part 25%
NH3, 1 part 28% H2O2, heat to 85° Celsius for 5
minutes, rinse with water) and TL2 wash (6 parts H2O, 1 part 37%
HCl, 1 part 28% H2O2, heat to 85° Celsius for 5 minutes,
rinse with water) to remove organic and inorganic substances and to make
them hydrophilic. Dry with N2. [0109]Put glass slide in a
vaporization chamber. Place 200 μl APTES in the chamber, vaporize at
60° Celsius for 10 minutes, bake at 150° Celsius for 60
minutes. Rinse in xylene and store in xylene. The amount of APTES might
be different for different vaporization chambers.

[0114]PTAA will be covalently bound to the surface in a thin layer, and
can be used to bind molecules of interest. Some modifications to the
protocol might be used, like concentrations, pH, buffers, incubation
times and materials used. The concentrations of EDC/NHS have been
selected to activate only a few of the carboxylic groups on the PTAA, to
increase the amount of PTAA bound to the surface and to preserve the
binding properties of the PTAA. Preferably only one side-group of the
polymer should be activated. It has not been evaluated if the reported
concentrations are optimal. The pH and the buffer composition used for
washing can be modified. In the protocol for Covalink microtiter plates
by Nunc the washing buffer is 116.9 g NaCl, 10 g MgSO4.7H2O,
0.5 ml Tween 20, 1 litre PBS. The incubation times can be modified to
improve the results. Sulfo-NHS can be used instead of NHS as it is
somewhat more stable.

Protocol, Immobilization of POMT

[0115]As POMT does not contain a carboxylic acid side-group, the procedure
for immobilization is reversed to that of PTAA. Instead of a APTES
surface a surface containing Carboxyl groups should be used, such as a
PEG matrix. The PEG matrix is treated with BrCH2COOH and NaOH to
introduce COOH groups, after which EDC/NHS is applied to the surface to
activate the carboxylic groups before POMT is added. Other surfaces could
also be used. The procedure has not been evaluated. See paper
"Photografted Poly(ethylene glycol) Matrix for Affinity Interaction
Studies", Biomacromolecules, Vol 8, No. 1, p. 287-295, 2007 for method
description.

Protocol, Immobilization of POWT and PTT

[0116]As POWT and PTT contain both a carboxylic and an amine group, using
EDC/NHS can cause crosslinking between polymer chains instead of between
the polymer and the surface, which is not desired. However, by tuning the
concentration of EDC/NHS and polymer, it might be possible to create a
thicker, crosslinked polymer layer covalently attached to the surface and
even better results. Either an APTES surface or a surface containing
Carboxyl groups could be used. The procedure has not been evaluated.

Surfaces

[0117]In the examples above a glass slide has been used for
immobilization. Other surface can also be used, such as silicon wafers,
glass beads, microtiter plates (Nunc's Covalink are pre-coated with an
APTES surface), or any other surface with the right properties.

Other Immobilization Techniques

[0118]Other immobilization techniques can also be used. For covalent
attachment, functionalized polymers with biotin or other terminal groups
can be used. For non-covalent attachment, physisorbtion to a protein
layer on a surface can be used. Polymer can for example be coated on Nunc
Maxisorp microtiter plates coated with dextran sulphate. Any other
techniques that will yield a polymer coated surface can also be used.

Adding Samples to the Surfaces

[0119]The sample to be evaluated or captured can be added to the polymer
coated surface in different manner. A volume of sample can simply be
added to the surface, incubated for an appropriate time and rinsed. Flow
canals can be used to lead the sample on to the surface in the proper
amount, which will yield a more repeatable system. If beads are used, the
can be added to the sample solution, incubate and be separated from the
solution using centrifugation, magnetic separation, or any other
separation technique.

Example 5

Lanthanide Nanoparticles Coated with Conjugated Polyelectrolytes

[0120]This example shows that a specific interaction between CPEs and
lanthanide nanoparticles, such as Eu2O3, Gd2O3 or
Nd2O3, is possible. These particles, which can be anything from
a few nanometer in size to a few hundreds of nanometer, might in turn be
useful for in vivo imaging of misfolded proteins and diseases related to
misfolded proteins according to the present invention. Modification of
the lanthanide particles is possible, such as doping with various metals,
such as Fe3+, Eu3+, Tb3+, etc. Before coating or binding
CPEs it is possible to pre-coat the lanthanide particles by for example
biomolecules, diethylene glycol, alkanes, carboxyl groups, citric acid,
amine groups etc before further adding other molecules.

[0121]To demonstrate the interaction between a few selected CPEs and
Gd2O3 nanoparticles the fluorescence of the CPEs was recorded,
see FIG. 13. Excitation wavelength was set to 400 nm and the CPE
measurement concentration was 2.5 μg/mL and the gadolinium oxide
particles was diluted 200 times from stock solution, bought from
Sigma-Aldrich. The measurement presented here was performed in double
distilled water but can be performed in many kinds of buffer solutions or
in a complex media.

[0122]All publications, patent applications, issued patents, and other
documents referred to in this specification are herein incorporated by
reference as if each individual publication, patent application, issued
patent, or other document was specifically and individually indicated to
be incorporated by reference in its entirety. Definitions that are
contained in text incorporated by reference are excluded to the extent
that they contradict definitions in this disclosure.

Patent applications by Fredrik Anghus, Stockholm SE

Patent applications by Olle Inganäs, Linkoping SE

Patent applications by Peter Åsberg, Stockholm SE

Patent applications by Peter Åsberg, Stockholm SE

Patent applications in class IN VIVO DIAGNOSIS OR IN VIVO TESTING

Patent applications in all subclasses IN VIVO DIAGNOSIS OR IN VIVO TESTING